A PHARMACOLOGICAL AND
TOXICOLOGICAL STUDY OF HEPTACHLOR
By
FRANK E. GREENE
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
February, 1962
ACKNOWLEDGMENTS
The author would like to express his sincere appreciation to
the members of his supervisory committee, Dr. Sidney Cassin, Dr.
Lauretta Fox, Dr. Melvin Fried, Dr. Thomas Malewitz, and Dr. Elbert
Voss, Chairman. Their encouragement, advice, and above all, patience
are gratefully acknowledged.
The author is indebted to the Graduate Council and the American
Foundation for Pharmaceutical Education for financial support which
made possible the completion of this study.
To his fellow graduate students the author extends special
thanks for their helpful suggestions, encouragement, and technical
assistance.
ii
I
•
•
•
TABLE OF CONTENTS
ACKNOWLEDGMENTS . ...... . . . .
Page
ii
iv
V
1
3
5
35
59
80
82
88
91
LIST OF TABLES . .
CHAPTER
III. TOXICITY STUDIES
IV. INVESTIGATION OF THE NATURE OF THL DIFFERENCE IN
SUSCEPTIBILITY TO THE ACUTE TOXIC EFFECTS OF HEPTACHLOR
IN MALE AND FEMALE RATS ................
V. LOCALIZATION OF THE SITE OF ACTION OF HEPTACHLOR . . .
VI. SUMMARY AND CONCLUSIONS
APPENDICES ....... . . . .
BIBLIOGRAPHY .
iii
LIST OF FIGURES
Figure Pa 8 e
1. EFFECTS ON THE GROWTH RATE OF VARIOUS CONCENTRATIONS OF
HEPTACHLOR IN THE DIETS OF MALE AND FEMALE RATS ...... 13
2. CONTROL LIVER, H AND E, 7 OX 17
3. LIVER OF FEMALE RAT FED 0.01 PER CENT HEPTACHLOR, H AND E,
90X • • 19
4. HIGH MAGNIFICATION OF CONTROL LIVER, H AND E, 350X .... 21
5. HIGH MAGNIFICATION OF LIVER FROM A FEMALE RAT FED 0.01 PER
CENT HEPTACHLOR, H AND E, 500X 23
6. LUNG OF CONTROL ANIMAL SHOWING CHRONIC INFLAMMATORY CHANGES,
H AND E, 100X 25
7. THE DOSE-PER CENT MORTALITY CURVE FOR HEPTACHLOR IN MALE
RATS 30
8. THE DOSE-PER CENT MORTALITY CURVE FOR HEPTACHLOR IN FEMALE
RATS 32
9. THE EFFECT OF SKF-525-A ON THE ACUTE TOXICITY OF HEPTACHLOR
IN MALE RATS 51
10. THE EFFECT OF THE CONTROL EMULSION ON THE BLOOD PRESSURE
AND RESPIRATION OF A PENTOBARBITALIZED DOG 63
11. THE EFFECT OF HEPTACHLOR ON THE BLOOD PRESSURE AND
RESPIRATION OF A PENTOBARBITALIZED DOG 64
12. THE EFFECT OF HEPTACHLOR AND ACETYLCHOLINE ON ISOLATED
RABBIT ILEUM . 67
CHAPTER I
INTRODUCTION
There is little information in the literature concerning the
toxicology and pharmacology of Heptachlor (1,4,5 ,6,7 ,8,8-Heptachloro-3a,
4,7,7a-tetrahydro-4,7 ,-methanoindene) . Frequently the information
required by law before an insecticide can be marketed is filed with the
United States Department of Agriculture and with the Food and Drug
Administration and never published (1). Other observations concerning
toxicity which may be made during field trials may wind up as progress
reports of some governmental agency where they are unavailable to a
literature searcher. Pharmacological studies may be undertaken later
when hazards of poisoning to warm-blooded animals become apparent,
pointing to the need for more basic knowledge of the mechanism of action
of the compound. With this information a rational approach to treatment
of accidental poisoning and to the development of safe means of
application of the compound by field personnel may be formulated.
In 1957, Heptachlor was selected as one of two pesticides to be
used in the fire ant extermination program undertaken in the Southeastern
United States. Shortly after initiation of this program it became
apparent that, in addition to its expected activity against insects,
the compound was highly toxic to wild and domestic animals.
Lack of knowledge of the pharmacologic effects produced by this
compound has forced those treating cases of accidental poisoning to
resort to empirical methods, which have not always been successful.
This investigation was undertaken in order to extend the infor-
mation concerning the pharmacologic and toxicologic properties of
Heptachlor. It was hoped that this additional information would lead
to a better understanding of the mechanisms of action of this compound.
To this end the activity of Heptachlor on the central and autonomic
nervous system and some factors influencing acute toxicity were studied,
CHAPTER II
THE CHEMISTRY OF HEPTACHLOR
Heptachlor, a member of the cyclodiene family of insecticides,
was first discovered as a constituent of technical Chlordan, under a
patent assigned to Hyman (2, p 60). Chemically, it is 1,4,5,6,7,8,8-
Heptachloro-3a,4,7 ,7a-tetrahydro-4,7 ,-methanoindene. The structural
formula was shown to be:
Other members of this family include Chlordan, Aldrin, and
Dieldrin whose structural formulas are given below. Endrin, a sterio-
isomer of Dieldrin; Isodrin, a sterioisomer of Aldrin; and Toxaphene,
a chlorinated camphene mixture, complete the family of cyclodiene
H CI
H CI
Chlordan
Heptachlor may be prepared by the action of sulfuryl chloride
on the condensation product of cyclopentadiene and hexachloro-cyclo-
pentadiene in carbon tetrachloride in the presence of benzoyl peroxide
(2, p 60).
All of the cyclodiene insecticides may be prepared by the
Diels-Alder reaction except Toxaphene, which is made by chlorinating
camphene to a chlorine content of 67 to 69 per cent (3, p 239).
Pure Heptachlor is a white crystalline solid, which has a
melting point of 95-96°C. The technical material available commercially
contains about 72 per cent Heptachlor and 28 per cent related materials
and is a soft, waxy solid, light tan in color, with a melting range of
46 to 74.9 C. Heptachlor is insoluble in water, but soluble in most
organic solvents (2, p 236). A recrystallized product is also available
for research purposes, which has a purity of about 90 per cent.
CHAPTER III
TOXICITY STUDIES
Review of the Literature
The available literature on the cyclodiene group of pesticides
which does not pertain to agricultural applications is concerned chiefly
with descriptions of toxic reactions occurring as a result of accidental
or experimental poisoning in animals. However, a few cases of fatal
poisoning in humans by Chlordan (4), Aldrin (5, p 5), and Toxaphene (6),
have been reported. The effects of these compounds on humans appear to
be identical with those observed in lower animals. No human fatalities
resulting from Heptachlor poisoning have been reported, but it must be
considered capable of producing severe toxic effects in humans if
precautionary measures are not used, as judged by reports of toxicity
to animals.
Gross symptoms of acute intoxication following a single exposure
are essentially the same for all members of the cyclodiene group of
pesticides. Among these symptoms are signs of central nervous system
stimulation which may progress to a series of clonic and tonic convulsions.
Evidence of increased activity of the parasympathetic nervous system is
usually present. Symptoms reported following acute Heptachlor poisoning
were increased salivation and lacrimation, generalized tremors, increased
respiratory rate and volume, violent clonic and tonic convulsions and
opisthotonus. In terminal stages, respiration was irregular with dyspnea
and cyanosis as prominent features (7).
By varying the size and number of doses, three separate types of
response to Dieldrin have been obtained (5, p 222). A few Large doses
resulted in one or more convulsions. Unless the animal died, there was
relatively prompt recovery without permanent damage or great weight loss.
Many doses of moderate size have produced a complete loss of appetite,
weight loss, and convulsions. Without treatment, death was seemingly
inevitable. Many small doses produced one or more convulsions without
any other apparent effect.
In addition to these effects, Aldrin (8) and Dieldrin (9) have
been reported to produce bradycardia, vasodepression, and miosis.
Lethargy and anorexia are common findings following Aldrin (10), Chlordan
(5, p 164), Dieldrin (5, p 226) and Endrin (11) poisoning. Partial to
total blindness, increased response to light tactle stimuli, and
convulsions induced by auditory stimuli have been attributed to Chlordan
poisoning (12). These effects have not been noted in cases of poisoning
by other members of this group.
It has been shown that absorption of these compounds through the
skin and mucous membranes in amounts sufficient to produce the toxic
symptoms previously described, is possible if the proper combination of
concentration and exposure time is provided (13).
Unpublished chronic feeding studies (14) have shown that rats
require about 10 parts per million (ppm) of Heptachlor in their diets
for production of tissue damage, when maintained at that level for 120
weeks. Such animals were reported to have manifested a slightly
depressed rate of growth, but their mortality was no greater than that
of the controls. However, females maintained on 30 ppm of Heptachlor
prior to mating had an increased rate of mortality of their offspring.
Animals given a diet containing 300 ppm died within eleven days, and
those given 100 ppm showed greater mortality than controls.
Pathological changes reported in the above study were: degenera-
tive changes in the cells of the central zone of the liver lobule, kidney
lesions involving degeneration of the epithelium of the proximal and
distal convoluted tubules, and some nonspecific changes in the neuronal
cells of the central nervous system. These effects are essentially the
same as those reported for other chlorinated hydrocarbons.
Toxicity data are greatly influenced by the specific experimental
conditions under which they were obtained. For this reason the results
of a particular experiment may be meaningless unless the exact experimental
conditions are also made known. This situation prevails particularly in
the literature on pesticides where the original data may lie in a file
of some governmental agency, with only the results, stripped of details
of experimental conditions, appearing in print. With this in mind, it
was considered advisable to determine the LDc n of Heptachlor under known
conditions.
Since there was such a pronounced sex difference in acute toxicity
of Heptachlor, it seemed likely that in chronic feeding experiments some
sex differences might also appear. This aspect seemed worthy of
investigation and rats of both sexes were maintained on diets containing
various amounts of Heptachlor for six months. This period of time was
suggested by Barnes and Denz (15), who felt that any pathology which will
develop with chronic exposure to a drug should appear within this period.
Experimental
Chronic Toxicity Studies
Materials and Methods
For chronic toxicity studies, immature rats between 50 and 100 Gms
were selected. Groups of ten rats of each sex were maintained on ground
Purina laboratory chow containing 1.0, 0.1, 0.01, and 0.001 per cent
Heptachlor. One group given ground lab chow alone served as controls.
The period of feeding was six months for the survivors. During this
period, the animals were given food and water ad libitum .
These rats were individually weighed and numbered at the onset
of the experiment. They were then weighed weekly for the first five
weeks, at the end of the seventh week, and again at the termination of
the experiment. At this time they were killed by stunning and exsanguin-
ation. In addition to the final body weights, organ weights were taken
for liver, kidneys, and gonads; and these data were analyzed statistically,
These organs, along with the lungs, adrenals, stomach, esophagus,
duodenum, colon, ileum, spleen, and heart, were taken for histological
examination. Rats which died in the course of the experiments were not
examined histologically as they were frequently mutilated by their cage
mates or were not found until a number of hours after death.
After fixation in 10 per cent formalin, all tissues, except those
used for frozen sections were dehydrated and infiltrated according to
a
Rats used in all experiments were NLR (Wistar origin) strain, supplied
by the National Animal Company, Creve Couer , Missouri.
b
Heptachlor used in these and all subsequent studies was supplied by the
Velsicol Chemical Corporation, Chicago, Illinois, and was labeled
"recrystallized" Heptachlor, 1009-17.
the butyl alcohol-paraf in mush method of Johnston et_ aJL. (16). The
parafin embedded tissues were sectioned at 8 microns and stained with
Harris hematoxylin and eosin as modified by Malewitz and Smith (17).
Liver sections were also stained for fat by the oil-red method
(18, p 124) and for glycogen by the periodic acid-Schiff (PAS) reaction
(18, p 132).
Results
All animals fed Heptachlor in concentrations of 1 or 0.1 per cent
were dead within two weeks. Body weights, taken at the end of the first
week of the experiment showed marked reduction in the growth rate of
these groups, particularly those on the 1 per cent diet. Symptoms of
toxicity began to appear for these two groups about the sixth day and
consisted chiefly of lethargy and anorexia with an occasional animal
showing signs of central nervous system stimulation manifested by
hyperexcitability and fighting among cage mates. About the eighth day
this stimulation became more apparent and convulsions of short duration
were seen frequently. Many of the animals developed lesions around the
muzzle as a result of fighting with their cage mates. The average
survival time of these groups is found in Table 1.
Average body weights taken for all groups are found in Table 2.
Examination of this table shows a slight depression of growth during
the first few weeks in the female groups fed the lower concentrations
of Heptachlor. Male rats fed 0.001 per cent Heptachlor also show a
slightly depressed rate of growth during the first two weeks. Growth
curves for the experimental groups surviving the entire period are
10
TABLE 1
AVERAGE SURVIVAL TIMES OF RATS'
FED HIGH CONCENTRATIONS
OF HEPTACHLOR
Diet
Sex
Average Survival
Time (Days)
17, Heptachlor
Male
10.6
17. Heptachlor
Female
9.7
0.17„ Heptachlor
Male
11.9
0.17. Heptachlor
Female
12.6
All groups contained ten animals,
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found in Figure 1. When comparing groups of the same sex, this graph
shows essentially parallel growth for the control and experimental
groups with the exception of the females maintained on the 0.01 per cent
Heptachlor diet. When t tests (19, p 164) were performed for the initial
and final body weights of all groups, this increase in weight was found
to be significant. Data obtained from statistical analysis of these
weights are found in Table 3. As a test for significance of all
comparisons made the confidence limits of 95 per cent probability were
arbitratily selected.
Three animals from the group of females fed the 0.01 per cent
diet died during the course of the experiment, one death occurring during
the fourth, and two during the sixth months of this study. All animals
from the other groups survived the entire experimental period.
On autopsy, no gross pathology was found in any of the organs
except the lungs, where occasional white nodules were seen. These
nodules were also found in the control animals and were considered to
result from pulmonary infection rather than exposure to Heptachlor.
Results of the statistical analysis of the liver, kidney, and gonad
weights are found in Table 4.
These data show that the livers of both male and female rats
from the 0.01 and 0.001 per cent groups differ significantly from the
controls. This difference in weight was an increase in all instances
except for the female group fed the 0.001 per cent diet, where this
change was a decrease. The kidneys of the 0.01 per cent male group also
show a significant increase in weight when compared with those of the
controls. The only significant change observed in gonad weights was an
increase found in the male group given 0.01 per cent Heptachlor.
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16
The most striking changes observed in all tissues studied were
found in the livers of female rats fed 0.01 per cent Heptachlor. The
cells of the peri-central vein region were greatly enlarged and pale
staining. Some cells had undergone karyolysis, while several multi-
nucleate cells were also seen. Margination of the cytoplasmic material
and enlarged nuclei were seen in many of the cells in this area. Photo-
micrographs of control and experimental livers showing these changes are
found in Figures 2, 3, 4 and 5. Cells of the peri-central vein region
contained neither fat, nor glycogen. Midzonal cells of sections showing
the cellular changes previously described showed dense glycogen
accumulation. In these same sections, fat droplets were prominent in
the peri-portal regions. These later areas were relatively glycogen poor.
These changes were present to a lesser degree in female rats fed
0.001 per cent Heptachlor and were seen only in the 0.01 per cent male
rat group. The observation of more extensive pathology in female than
in male rat livers offers a possible explanation for the greater number
of deaths which occurred in the female group fed 0.01 per cent Heptachlor.
The lungs of practically all animals, experimental and controls,
showed evidence of chronic infection. Congestion, lymphocytic infiltra-
tion, and areas of necrosis were present in varying degrees in all
animals. Hemosiderin engorged cells, indicative of chronic congestion
were prominent in many lungs (20, p 56). A photomicrograph showing some
of these changes is found in Figure 6.
The author would like lo express his appreciation to Drs. J. E. Edwards
and M. Waid of the Department of Pathology, J. Hillis Miller Health
Center, for their valuable suggestions and interpretations of the
pathology found in the livers of these animals.
FIGURE 3. LIVER OF FEMALE RAT FED 0.01 PER CENT
HEPTACHLOR, H AND E, 9 OX
20
»
FIGURE 5. HIGH MAGNIFICATION OF LIVER FROM A FEMALE RAT
FED 0.01 PER CENT HEPTACHLOR
H AND E, 500X
<
24
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v
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•
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-* ■ ^^k ^^^^^A • mm* '
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FIGURE 6. LUNG OF CONTROL ANIMAL SHOWING CHRONIC
INFLAMMATORY CHANGES, M
H AND E, 100X ^
i
26
27
The results of histological examination of the other tissues and
organs were non-remarkable.
Acute Toxicity Studies
Determination of the Oral LD50 of the Corn Oil Solution of Heptachlor
Materials and Methods
Preliminary investigation had shown the dosage range for to
100 per cent fatalities in male rats to be from 40 to 100 mg/Kg, and in
females from 60 to 190 mg/Kg. Doses at graded increments were administered
orally to groups of eight rats each using a Phipps and Byrd oral needle
following a period of fasting of 12 to 18 hours. The rats had free
access to water during the period of fasting and were given food
immediately following the administration of Heptachlor. These rats were
then observed for a period of eight days, and the LD50 was calculated
from the number of deaths occurring during this period using the method
of Litchfield and Wilcoxon (21).
The general procedure as outlined by this method is followed
in the calculation of the LD50 f° r tne male rats found in Appendix III.
For determination of the female LDcq, only the results are shown.
Results
Deaths resulting from a single oral dose of Heptachlor usually
fell into two categories: 1) those dying within the first 24 hours,
and 2) those dying from about the fifth to the eighth day. These
intervals represent the period of early and delayed toxicity respectively.
The method used for the preparation of the corn oil solution is found
in Appendix I.
28
In both periods the symptoms prior to death were similar and consisted
of a series of short spasms which appeared in increasing frequency,
ultimately terminating in a clonic and tonic convulsion. Following these
convulsions, the muzzles of all animals were wet, indicating that an
increase in secretion had occurred during the seizure. This finding was
indicative of parasympathetic nervous system stimulation, and was seen
following convulsions only. The LJ^o's and their confidence limits are
given below.
Male rats .- The data obtained from the determination of the LD50
are given in Table 5. The graph for the data is found in Figure 7. The
LD5O and its 19/20 confidence limits calculated from these data is 59
(49 to 71) mg/Kg.
Female rats .- The data obtained from this experiment are found
in Table 6, and presented graphically in Figure 8. The calculated LD50
and its 19/20 confidence limits is 132 (114 to 154) mg/Kg.
Discussion
The LD50 values determined for male rats are in reasonable
agreement with those previously reported (14). The periods of early and
delayed toxicity as described by Lehman (13) were also seen in this study,
As in previous studies (14) male rats proved to be much more susceptible
to a single oral dose of Heptachlor than females. The nature of this
difference is investigated in the following chapter.
The difference in susceptibility of male and female rats was
reversed in the acute and chronic toxicity studies. While males were
more susceptible to the acute effects of Heptachlor, females were more
29
»
TABLE 5
SOLUTION OF THE DOSE MORTALITY CURVE OF THE CORN OIL
SOLUTION OF HEPTACHLOR IN MALE RATS
>
Observed*
Expected
Observed
Contributions
Dose
Killed
Per Cent
Per Cent
Minus
t0 2
(Chi) Z
mg/Kg
Tested
Mortality
Mortality
Expected
40
0/8
4.4
13
8.6
0.060
50
1/8
12.5
34
21.5
0.190
60
4/8
50.0
52
2.0
0.002
70
5/8
62.5
68
5.5
0.025
80
7/8
87.5
81
6.5
0.028
100
8/8
98.4
93.5
4.9
0.039
>
The observed values listed for and 100 per cent effect
represent corrected values obtained by the Litchfield and Wilcoxon
method.
The expected values were obtained from Figure 7.
30
99.9
" 1 ' | ' | ' ' | ' ' ' '
99
o /
>
95
- / -
90
/ —
>-
/
H
(y
_i
- I —
<
I
t-
1
<r
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o
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r
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5 50
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1
o
cr
UJ
1
a.
/ °
10
~ —
>
I
/ °
• 1 i 1 i 1 i i 1 i i i i
20 30 50 70 100 200
DOSE, MG/KG
Fig. 7. --The dose-per cent mortality
curve for Heptachlor In male rats.
31
TABLE 6
SOLUTION OF THE DOSE MORTALITY CURVE OF THE CORN OIL
SOLUTION OF HEPTACHLOR IN FEMALE RATS
Observed
b
Expected
Observed
Contributions
Dose
Killed
Per Cent
Per Cent
Minus
t0 2
(Chi) Z
mg/Kg
Tested
Mortality
Mortality
Expected
60
0/8
0.6
1.8
1.2
0.008
80
1/8
12.5
9.0
3.0
0.011
100
1/8
12.5
22.0
9.5
0.050
120
2/8
25.0
39.0
14.0
0.080
140
4/8
50.0
55.0
5.0
0.010
150
7/8
87.5
62.0
25.5
0.275
160
6/8
75.0
70.0
5.0
0.011
170
8/8
92.6
74.0
18.6
0.180
The observed values listed for and 100 per cent effect
represent corrected values obtained by the Litchfield and Wilcoxon
method.
'The expected values were obtained from Figure 8.
32
95
ii ii i i i i I
90
^_ / ___
1
/
o/
50
— >0 —
>-
_)
cr
o
2
/ °
1
l-
O/0
^ 10
— /
PERI
i 1 i . , i
0.1
— / —
>
'
0.01
1 1 — i — 1 i i 1 i i i i 1
40 70 100 200 300
DOSE, MG/KG
Fig. 8. — The dose-per cent mortality
curve for Heptachlor in female rats.
33
susceptible to chronic exposure to Heptachlor. The only pathologic
differences observed in the surviving animals was a more extensive liver
damage in female rats, when compared to males fed the same concentration
of Heptachlor. If the death of the three animals in the female group
fed 0.01 per cent Heptachlor resulted from this type of damage, it is
possible that the lower mortality rate observed in male rats fed the
same diet could be due to some protective action of this organ by
testosterone. This protective role of testosterone has been proposed
by Seyle (22). The significant increase in the weight of the testes of
rats fed 0.01 per cent Heptachlor could then represent a functional
response to an increased utilization of testosterone. This event could
arise following an increased demand of long duration for this hormone
if it should be involved in such a role in the liver.
The over-all activity of Heptachlor falls into two, apparently
unrelated, categories: 1) central nervous system stimulation seen in
acute poisoning with a single large dose, or frequently administered
smaller doses, and 2) pathological changes in organs such as the liver
and kidney, following continuous exposure to small amounts of this
compound. The period of delayed toxicity may contain elements of both
types of action.
The anorexic effect of Heptachlor and the property of lipids of
the central nervous system to resist mobilization in the face of
protracted starvation suggested the following sequence of events in the
period of delayed toxicity of Heptachlor.
Following ingestion of Heptachlor in an amount less than the
quantity necessary to cause death in the early toxicity period,
34
Heptachlor and the epoxide are stored in body fat. During the period of
anorexia which may develop, the animal will mobilize this fat when
glycogen deposits are depleted, but lipids of the central nervous system
would not be involved in this process. Heptachlor, which is stored in
body fat would be mobilized as the fat in which it is stored is broken
down. The Heptachlor displaced in this manner would tend to relocate
in other lipid tissue. The unmobilized lipid of the central nervous
system is a likely site of such events. Through such a process Heptachlor
concentration in the nervous system could gradually build up, leading to
the symptoms of central nervous system stimulation and ultimately,
convulsions in these animals.
A logical treatment of Heptachlor poisoning should be directed
toward the two phases of the action of Heptachlor: 1) control of the
convulsions during the period of early toxicity, and 2) prevention of
the secondary toxic effects. Control of the later effects could possibly
be accomplished by supportive therapy directed toward protection of the
liver and other organs from further damage. A low fat, high protein
diet, plus one or more lipotropic substances (23, p 928), is recommended
for treatment of damaged livers, and would, at the same time provide
nutritional support for the animal. Such a regimen would be expected
to prevent mobilization of body fat and secondary deposition of Heptachlor
in the central nervous system, if this event should actually take place.
The central nervous system effects could probably be controlled by
cautious administration of barbiturates.
CHAPTER IV
INVESTIGATION OF THE NATURE OF THE DIFFERENCE IN
SUSCEPTIBILITY TO THE ACUTE TOXIC EFFECTS OF
HEPTACHLOR IN MALE: AND FEMALE RATS
Review of the Literature
In the course of work directed toward determination of tissue
residues from Heptachlor, Davidow and Radomski (24) found a metabolically
altered derivative, an epoxide, along with Heptachlor in the fat of
Heptachlor-f ed dogs. They reported the following as the structure of
this compound:
H CI
These authors stated that this was a type of biological oxidation product
which has not previously been reported, and suggested that it may be a
stable intermediate of the process of biological hydroxy lation which,
due to its solubility in fat, tended to accumulate in adipose tissue.
Since this initial discovery, the epoxide of Heptachlor, along with the
parent compound, has been isolated and identified in the body fat of
rats and rabbits (25), (26), and in the whole insect in the case of the
housefly, Musca domesticus (27). The female rat stores the epoxide to
a much greater extent in fat than does the male, the biological
35
36
a
multiplication ratio being 6.2 for the female and 1.2 for the male
when both are maintained on 30 ppm of Heptachlor in their diet (25).
A particularly interesting aspect of the toxicity of Heptachlor
in the rat is the difference in response of the male and female to a
single oral dose, the reported LDcq for females being 142 mg/Kg, and
that for males, 60 mg/Kg (14). A possible explanation for this differ-
ence could be a different rate of conversion of Heptachlor to its
epoxide, a compound which has an intravenous toxicity distinctly greater
than the parent compound. When given a dose of 10 mg/Kg, 100 per cent
of epoxide treated mice died, while Heptachlor at the same dosage level
caused no deaths (25). If male rats were capable of converting Heptachlor
to its epoxide at a faster rate, this might explain the higher mortality
produced in males than females given the same dose.
Similar differences in sex responses have been observed with
other compounds, particularly the barbiturates, in which it was noted
that male rats consistently slept for shorter times than females given
the same dose of pentobarbital. Jarcho (28) noted that this sex
difference could be demonstrated only with the barbiturates which are
known to be detoxified in the liver. Hoick et a_l. (29) investigated
the effects of testosterone propionate and estradiol dipropionate
administered prior to hexobarbital treatment in normal male and female
rats and was able to shorten the sleeping time of testosterone-treated
females to that of the males. They also were able to reduce the sleeping
The biological multiplication ratio is a measure of the ability to
concentrate a substance in the body fat and is calculated by dividing
the concentration of the substance in the diet into the concentration
of the substance found in the fat.
37
time in the testosterone-treated male below that of the nontreated
control males. Cameron e_t_ a_l. (30) reported an increase in mean
barbiturate-induced sleeping time following castration in male rats.
Tureman et al_. (31) found that gonadectomy increased sleeping time for
both sexes, and that testosterone given to male gonadectomized rats and
intact females decreased sleeping time. Changes in sleeping times have
provided a convenient method for the determination of the effects of
various experimental procedures on metabolic transformations which
resulted in the inactivation of certain barbiturates. Conditions which
produce changes in sleeping time may also be expected to affect the
rate of biotransformation of other substances whose metabolism does not
provide so convenient an endpoint .
Quinn et_ a_l. (32) investigating the biotransformation of
hexabarbital by the microsomal fraction from rat liver homogenate, found
close correlation between the rates of biotransformation and the sleeping
time. Female rats slept four to five times as long as males and showed
a correspondingly lower rate of biotransformation. Further, this
difference in the sexes was reduced by administration of testosterone
to females for six weeks prior to testing, producing both an increase
in biotransformation and a corresponding shortening of sleeping time.
In addition to these drugs, sex differences in the rates of
metabolism in rats have been noted with two non-chlorinated hydrocarbon
insecticides, Schradan and Parathion (33). These compounds are
relatively weak cholinesterase inhibitors in vitro , but are converted
in vivo to powerful cholinesterase inhibitors, Schradan was found to
be more toxic for males and Parathion for females, The liver of the
38
male rat has been shown to convert Schradan to its more active oxide
better than the livers of females; the reverse holds true for Parathion,
which is converted to paraoxone by the liver of the females more rapidly
than in that of the male. These reactions were found to occur in the
microsomal fraction of liver homogenate and to be DPN and Mg dependent.
Male and female rats are equally susceptible to the cholinesterase
inhibitor TEPP, which does not undergo conversion in_ vivo .
Hoick et_ al_. (34) produced hypothyroidism in rats of both sexes
by using 200 mg of propylthiouracil mixed with 1 Kg of food. The animals
were maintained on this diet for 20 days prior to testing. This
experimental hypothyroidism was expected to decrease liver metabolism
and thereby prolong the action of those barbiturates metabolized in
this organ. Using pentobarbital, he was able to produce an increase in
sleeping time in both sexes, but a significant difference between the
male and female response was still present.
The Smith, Kline and French Laboratories have synthesized a
compound which was found to be capable of greatly increasing the effects
of certain drugs by affecting the rate at which they are degraded in the
body. This compound, beta-diethylaminoethyl-diphenylpropyl acetate, was
given the generic name of diphenylpropylacetate and referred to as
SKF-525-A. It was sent to the National Institutes of Health where its
effects on the biotransformation of a variety of drugs were studied.
Axelrod e_t_ al. (35) testing the effects of pretreatment with SKF-525-A
on hexabarbital sleeping time, found it to be increased four times over
that of the saline-treated controls. He also followed blood levels of
the barbiturate during this period and found the half-life paralleled
39
this increase. Blood levels of hexobarbital were measured in animals
at the time they regained their righting reflex and were found to be the
same regardless of whether the animal had been pretreated with SKF-525-A
or not. In addition, animals recovering from anesthesia cannot be
reinducted by use of SKF-525-A as is the case with chlorpromazine or
glucose, which are classified as potentiators (35). On the basis of
these findings, SKF-525-A was judged to be a "prolonging" agent rather
than a potentiator.
Cook et al. (36) determined that the maximum effect of hexobarbi-
tal sleeping time was obtained when SKF-525-A was given 40 minutes prior
to administration of the drug. He also found SKF-525-A to be equally
effective when given either intraper itoneally or orally. He was able
to produce a 35-fold increase in the sleeping time of rats pretreated
with SKF-525-A over saline treated controls. In other studies, Cook
et_ al. (37) found that SKF-525-A had no effect on thiopental, ether, or
nitrous oxide anesthesia, or on the sleeping times of barbital, or
methylparafynol. None of these substances are inactivated by metabolism.
However, SKF-525-A did enhance the analgesic properties of morphine
sulfate, codeine phosphate, and methorphinan, and prolonged sleeping
times of Seconal, Amytal, Butethal, Ortal, pentobarbital and chloral
hydrate. Metabolism of members of the later group results in loss of
activity.
In vitro studies by Cooper (38), using liver homogenates
demonstrated the inhibition of a variety of biotransformation reactions
with SKF-525-A. Some of these were: side chain oxidations, dealkylations ,
deaminations and cleavage of ether linkages. La Du (39), investigating
40
a group of compounds whose biotransformations could be accomplished
with a system comprised of liver microsomes plus reduced TPN and oxygen,
found that all could be inhibited by SKF-525-A. The conversion of
Parathion and Scaradan to their oxides, cited previously (33), and
found to be DPN dependent was also inhibited by SKF-525-A.
Cooper (40) tested the effects of SKF-525-A on enzyme systems
involved in generating reduced TPN and in transporting hydrogen from
reduced TPN to oxygen via the cytochrome system. He also tested effects
on DPN-requiring enzymes, using the alcohol dehydrogenase system. He
was unable to demonstrate inhibition in either case and concluded that
the action of SKF-525-A in_ vivo probably lay in its effect on some
other, as yet unidentified, system common to all of the biotransformation
reactions studied.
Since the discovery of these properties of SKF-525-A, two other
compounds have been found which inhibit the same biotransformation
reactions. They are 2 ,4,-dichloro-6-phenoxyethyl diethylamine (Lilly
18947) and iproniazid. Studies on the effects of these compounds on
hexobarbital sleeping time by Fouts and Brodie (41), (42) have shown
Lilly 18947 to be most effective, followed by SKF-525-A and iproniazid,
in that order.
Recently, workers at the Lilly Laboratories have synthesized
another compound designated as Lilly 32391 (2 phenyl-4,6-dichloro-phenoxy)
ethylamine HCl, which is said to be about ten times as potent as Lilly
compound 18947 (43). The structures of these compounds are shown in
Table 7.
41
TABLE 7
STRUCTURAL FORMULAS OF SOME INHIBITORS
OF DRUG METABOLISM
/? / C 2 H 5
C-0-CH 2 -CH 2 -N
C \ X °2 H 5
V CH 2 -CH 2 -CH 3
0=C-NH-NH-CH-CH,
CH,
t S>
SKF-525-A
Ipronlazld
^^
•HBr
92 H 5
l-f VO-CH 2 -CH 2 -N-C 2 H 5
^^
CI-/ Vo-CH 2 -CH 2 -NH 2 -HCI
Lilly Compound 18947
Lilly Compound 32391
42
Metabolic activation of Heptachlor rather than detoxification is
strongly suggested by data from the experiments previously discussed
concerning the nature of sex difference in response to certain barbitu-
rates. Since male rats are capable of metabolizing barbiturates at a
faster rate than females, it is not unreasonable to assume that similar
differences in the metabolism of other compounds may exist. If Heptachlor
falls into this category, the difference in response by male and female
rats could be explained by a more rapid rate of conversion, in the male,
to a compound more toxic than the original one. The only known metabolite
of Heptachlor, its epoxide, is in fact more toxic than the parent
compound. In view of these facts, it was felt that investigations
analogous to the studies of sex differences in barbiturate response
could yield valuable information concerning some of the factors involved
in the mechanism of the toxic action of Heptachlor.
Experimental
The Effect of Gonadectomy and Gonadal Hormones on Acute Toxicity
Materials and Methods
To determine the effects on the acute toxicity of Heptachlor
produced by castration and the administration of exogenous hormones to
castrated animals, thirty male and thirty female rats weighing 150 to
200 Gms were castrated by the methods of D'Amour and Blood (44, p 44, 45)
and allowed one month to recover. At this time, the separate groups of
males and females were each divided into three sub-groups of ten animals;
and to these, one normal (uncastrateu) group of each sex was added for
controls.
43
Daily hormone or sesame oil injections were given subcutaneously
according to the schedule given below. These injections were begun two
weeks before and continued one week following administration of
Heptachlor. The dosage regimen selected was one used by Hoick et al. (29)
in their studies on sex differences in pentobarbital sleeping time in
rats.
Group Treatment Dosage
Non-castrate Sesame oil 1 ml/Kg/day
Castrate Sesame oil 1 ml/Kg/day
Castrate Estradiol dipropionate 1 mg/Kg/day
Castrate Testosterone propionate 1 mg/Kg/day
On the fourteenth day of treatment, the rats were weighed and
given an oral dose of 200 mg/Kg of Heptachlor. These rats were older
and heavier than those used in the determination of the LDcq, and
preliminary trials had shown them to be more resistant to the toxic
effects of Heptachlor than younger rats. These trials had indicated
that at a dosage level of 200 mg/Kg, one could expect about 80 per cent
fatality in males and 40 per cent fatality in females. The animals
were observed for fourteen days following administration of Heptachlor
and all deaths were recorded. Dead animals were autopsied, and the
seminal vesicles of the males, and uteri of the females, were removed
and examined for gross effects. These organs were not examined
histologically.
The testosterone used in these studies was manufactured by Charles F.
Pfizer and Company, Brooklyn, New York, under the trade name Synandrol
(lot no. 88354). Estradiol was supplied by Ciba Pharmaceuticals
Incorporated, Summit, New Jersey, as their trade name product, Ovocylin
(control no. 242156).
44
Results
The data from this experiment are found in Table 8. The expected
sex difference appeared in the non-castrate group with twice as many
males as females dying during the first day. Testosterone increased
the toxicity of Heptachlor for castrate males and females to the level
of normal males and shortened the length of time necessary for symptoms
of toxicity to appear. All estradiol-treated animals escaped the early
period of toxicity, but some deaths did occur in both male and female
rats beginning on the fourth day. At the end of the first week, the
sesame-oil-treated, castrate animals showed the same malerfemale death
ratio as the non-castrate, sesame-oil-treated animals; although the
actual number of fatalities was less in the former group. During the
second week, several fatalities occurred in the castrate sesame-oil-
treated and in the castrate, estradiol-treated groups. These deaths
were delayed longer than the expected period of delayed toxicity which
usually lasts from about the fourth to the eighth day. In this
particular experiment, however, this period was very mild for the
control animals with only an occasional animal showing signs of central
nervous system stimulation. No deaths occurred among the control groups
during this period. By the end of the second week, the total number of
deaths was essentially the same for all groups except the non-castrate,
sesame-oil-treated female group. The latter group had the lowest number
of fatalities during the experimental period.
All animals receiving testosterone or estradiol lost weight
during the two week period prior to the administration of Heptachlor.
Sesame-oil-injected animals showed a slight increase in body weight
45
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46
during this period. These changes will be tabulated along with the
weight changes of other experimental groups at the end of the chapter.
The Effect of Hormone Administration on Acute Toxicity
of Heptachlor in Normal Rats
Materials and Methods
This experiment was undertaken in order to determine if the
effects observed in castrate animals could be demonstrated in normal
animals. An additional point of investigation was to determine if
Nilevar (17-ethyl-19-nortestosterone) had any demonstrable effect on the
toxicity of Heptachlor. Nilevar is considered to be an anabolic steroid
as it has an anabolic : androgenic ratio of 20; whereas this ratio for
testosterone is about one (45, p 893). This point of investigation arose
when it was observed in the previous experiment that testosterone greatly
increased the toxicity of Heptachlor in castrate rats. If this activity
was due to an anabolic rather than androgenic effect, Nilevar could be
expected to increase toxicity, also, as it shares the anabolic activity
of testosterone while having only a fraction of its androgenic activity.
The experimental groups and their dosage regimen is listed below:
Group Treatment Dosage
I Sesame oil 1 ml/Kg/day
II Estradiol dipropionate 1 mg/Kg/day
III Testosterone priopionate 1 mg/Kg/day
IV Nilevar 3 1 mg/Kg/day
The same series was set up for each sex, and each group contained
8 animals. The injections were given subcutaneously every day according
a The Nilevar (control no. 2041) used in this experiment was manufactured
by G. D. Searle and Company, Chicago, Illinois. The other hormone
preparations were the same as those used in the previous experiment.
47
to the schedule previously listed. They were given two weeks before
and one week after 200 mg/Kg of Heptachlor was given orally. These
animals were observed for a period of two weeks after the administration
of Heptachlor, and deaths were recorded as they occurred. Dead animals
were autopsied, and gonads and accessory sex organs were removed and
examined grossly for effects produced by their particular treatment.
Results
When compared with controls, the data from this experiment, found
in Table 9, do not show a clear-cut effect resulting from the administra-
tion of hormones to normal animals. Group I, the sesame-oil-treated
controls, showed the normal pattern of male and female toxicity; but
the delayed interval of the acute toxicity period occurred later than
usual in the female group.
Estradiol appeared to have increased the early toxicity of
Heptachlor in male rats. Deaths due to delayed toxicity developed more
rapidly in females treated with this hormone than in the sesame-oil-
treated group.
During the first day mortality was much higher in the testoste-
rone-treated males than in other groups. Four animals from this group
died; while only one death occurred in all other groups combined. At
the end of the experimental period, all animals from this group were
dead. Toxic symptoms appeared sooner in the testosterone-treated
females than in the controls. Symptoms of the former group began on
the third day and lasted until the tenth day.
The pattern of toxicity of Heptachlor in male rats which were
pretreated with Nilevar differed from that of other experimental groups.
48
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Normally, most of the fatalities which will occur in a group of animals
given a standard dose of Heptachlor will fall in a two or three day
period. Nilevar-treated animals, however, did not follow this pattern.
Deaths occurred in this group from the first through the tenth day
following Heptachlor. Mortality of females given this hormone before
Heptachlor was slightly lower than in the female control group,,
Gross examination of the gonads and accessory sex organs of
those animals which died during the course of the experiment showed a
consistent enlargement and fluid distention of the seminal vesicles of
the testosterone-treated males and atrophy of these structures in animals
receiving estradiol* Uteri of estradiol-treated animals exhibited
increased vascularity when compared to controls; while uteri from the
testosterone-treated groups were uniformly pale. No effects on either
of these tissues were seen in the Nilevar-treated animals.
Changes in body weight will be tabulated along with the weight
changes of other experimental groups at the end of this section.
The Effect of SKF-525-A on the Acute Toxicity
of Heptachlor in Male Rats
Materials and Methods
This experiment was undertaken in order to test the hypothesis
that Heptachlor was metabolized to a more toxic product; and if this
metabolism could be slowed or stopped, toxicity would be decreased.
SKF-525-A was selected for this experiment because of its known ability
to inhibit the metabolism of a variety of compounds.
SKF-525-A was supplied by the Smith, Kline and French Laboratories,
Philadelphia, Pennsylvania.
50
Twenty-four, 150 to 200 Gm male rats were divided into three
groups of eight rats each and were given the following treatment:
Groups Treatment
A Water 10 ml/Kg
B SKF-525-A 100 mg/Kg
C SKF-525-A 100 mg/Kg every twelve hours
This treatment was given orally about forty minutes before the oral
administration of 150 mg/Kg of Heptachlor, This period of time was
chosen because SKF-525-A has been shown to give maximum prolongation of
hexabarbital sleeping time forty minutes after ingestion (36). Since
this same work had shown SKF-525-A to be active as long as fifteen hours
after administration, one group was given this compound every twelve
hours to determine if additional benefits could be gained by multiple
dosage. All animals were observed for a period of eight days, and
deaths were recorded.
Results
The graph for the data from this experiment is found in Figure 9.
Prior treatment of SKF-525-A was shown to produce a marked delay of the
onset of toxic symptoms when compared with water-treated controls.
Examination of the graph shows that some additional protection was
obtained by administration of SKF-525-A every twelve hours. Five
animals from the singly treated group died after the first twenty-four
hours; while only two deaths occTurred in the group given SKF-525-A
every twelve hours.
51
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52
The Effect of Propylthiouracil (PTU) on Acute Toxicity
Materials and Methods
This experiment was undertaken in an effort to determine if PTU
induced hypothyroidism, and resulting decrease in metabolic activity (34)
would effect the acute toxicity of Heptachlor in rats. Twenty-four
animals of each sex were placed in three groups of eight rats each,
body weights recorded, and two groups of each sex were given 0.15 per
cent PTU in ground Purina laboratory chow ad libitum for a period of
three weeks. As a control, the remaining group of each sex was fed
ground laboratory chow, alone. At the end of this period, the animals
were weighed, and one group of the PTU fed and the control fed group
from each sex was given Heptachlor 200 mg/Kg orally The remaining PTU
group from each sex was given corn oil, 5 ml/Kg and observed for possible
toxic effects which might have been produced by PTU. After administration
of Heptachlor, the animals were observed for a period of eight days, and
deaths were recorded „ At death, the animals were autopsied and the
thyroid glands removed and examined grossly for the effects of PTU.
Results
Toxic symptoms developed rapidly in both male and female rats
which had been maintained on PTU prior to the administration of Heptachlor.
All males from these groups were dead within one and one-half hours.
Females from the PTU-Heptachlor group developed symptoms of central
nervous system stimulation within one hour, and seven were dead in less
than twenty-four hours.
a The propylthiouracil used for this experiment was obtained by crushing
50 mg tablets (control no. Y 563J) manufactured by Parke, Davis, and
Company, Detroit, Michigan.
53
Animals fed regular laboratory chow developed symptoms more
slowly, although all males from this group died within twenty-four hours.
Only two rats from the female group given regular laboratory chow died
within the eight-day observation period. No deaths occurred in the
groups fed the PTU diet and given corn oil.
The thyroids of PTU-treated animals which died during this
experiment appeared to be greatly enlarged when compared to non-PTU-fed
controls. All male rats, including controls, lost weight during the
experimental period. Both groups of females given PTU in their diet
showed a weight reduction, while female rats given the control diet
gained weight. Changes in body weight will be tabulated along with the
weight changes of other experimental groups at the end of this section.
Discussion
Evidence of a possible endocrine basis for the difference in
sex response is presented in Table 10. Castration decreased the number
of early deaths in both male and female rats. However, at the end of the
experimental period, there was little difference between these groups and
the non-castrate males which characteristically demonstrate the highest
percentage mortality for a given dose of Heptachlor. Data in this table
show only slight differences between any experimental groups at the end
of the two week period of observation. Normal females still had fewer
deaths than other groups, except for females given Nilevar. The latter
group had one less fatality than the control females.
The rate at which the toxic effects of Heptachlor appeared was
accelerated in both castrate and normal animals treated with testosterone.
54
TABLE 10
COMBINED DATA FROM EXPERIMENTS CONCERNING THE
ENDOCRINE INFLUENCE ON THE ACUTE
TOXICITY OF HEPTACHLOR IN RATS
Number of Deaths on Day Indicated
Following Oral Administration
of Heptachlor, 200 mg/Kg
(
Jroup
Killed
Tested
Per Cent
Killed
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Female
I
a
Normal
2
2
1
5/8
62.5
II
Normal
2
2
1
1
1
7/8
87.5
III
Norma 1
1
1
1
1
1
1
6/8
75.0
IV
Normal
2
1
1
4/8
50.0
V
Norma 1
3
1
4/8
50.0
VI
_ a
Castrate
1
1
2
2
1
7/8
87.5
VII
Castrate
1
2
1
1
2
7/8
87.5
VIII
Castrate
7
7/8
87.5
Mai
e
I
a
Normal
4
3
7/8
87.5
II
Norma 1
1
5
6/8
75.0
III
Normal ,
Normal
4
3
1
8/8
100.0
IV
2
1
1
1
2
7/8
87.5
V
Normal
6
6/8
75.0
VI
Castrate
Castrate
2
1
1
2
1
7/8
87.5
VII
1
2
1
1
1
6/8
75.0
VIII
Castrate
6
1
7/8
87.5
a Plus Sesame Oil, 1 ml/Kg/day
b,
Plus Estradiol Dipropionate, 1 mg/Kg/day
Plus Testosterone Propionate, 1 mg/Kg/day
Plus Nilevar, 1 mg/Kg/day
55
This effect was prominent in castrate male and female rats, while in
normal rats this response was not as well defined. These observations,
together with a slower development of toxicity in the castrate male,
suggest that an active role is played by the gonadal hormones of male
rats in the acute toxicity of Heptachlor. An additional observation
favoring this explanation of the role of male hormones is the significant
increase in the testicular weight of male rats fed 0.01 per cent
Heptachlor, presented in Chapter III. Since no pathological changes
were found in these organs, this increase in weight could have resulted
from a physiological hypertrophy caused by an increased demand for
gonadal hormones. Such a phenomenon could result from continuous demand
for these hormones in the metabolism of Heptachlor. A change in weight
has not been previously reported following chronic exposure to Heptachlor,
Estrogenic effects on the toxicity of Heptachlor appear to be
inconsistent. Castration increased the number of deaths in female rats,
but estradiol administered to castrate females failed to prevent this
increase in mortality.
Changes in body weight observed in the experimental animals from
both hormone experiments along with changes which were recorded for
propylthioracil-treated animals are listed in Table 11. It was observed
that some of the procedures used in these experiments produced a weight
loss while other procedures increased body weight. A weight change
brought about by a change of the size of the lipid depot of the body
could alter the space available for storage of Heptachlor and its
metabolite. This, in turn, could effect the susceptibility of an animal
to this compound. However, comparison of the weight changes and the
56
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number of mortalities observed in all groups did not show any relation-
ship between weight changes and toxicity.
The delaying effects of SKF-525-A on the appearance of toxic
symptoms provides support for the theory of metabolic activation of
Heptachlor. It is difficult to assess the actual benefit obtained by
repeated administration of SKF-525-A, due to a higher initial number of
deaths in the group which was given this compound every twelve hours.
As a result of this there were fewer animals to observe in this group
later in the experiment than in the group which only received a single
treatment of SKF-525-A.
The opposing effects of SKF-525-A and testosterone on the
metabolism of certain drugs, as demonstrated by hexobarbital sleeping
time studies, were also seen during these experiments. The effect of
these compounds were reversed in their influence on Heptachlor toxicity,
as testosterone increases toxicity while SKF-525-A delays or decreases
toxicity. The opposite holds true for activity of hexobarbital, which
is inactivated by metabolic pathways. In the later case, testosterone
shortens the activity, while SKF-525-A prolongs the effects of this
drug. Since inhibition of drug metabolism has not been reported for
testosterone, nor stimulation of this process by SKF-525-A, it may be
proposed that testosterone, by increasing the rate of biotransformation
to the epoxide, increases the toxicity of Heptachlor, while SKF-525-A,
through its inhibitory action on this reaction, delays the appearance
of toxic symptoms. One point which must not be overlooked, however, is
the probability that, only the rate of metabolism is effected by these
compounds, and that some metabolic conversion of Heptachlor will occur
58
regardless of the endocrine condition of the animal. In addition, there
is no evidence that Heptachlor per se is inactive, and it is quite
possible that the toxic effects observed in a particular animal at a
given time are produced by the sum of the concentrations of both Heptachlor
and its epoxide present at that time.
The action of propylthiouracil in speeding the onset of toxic
reactions and increasing the susceptibility of rats to Heptachlor was
unexpected, since PTU induced hypothyroidism normally slows metabolism,
thereby prolonging pentobarbital sleeping time in rats (34), an effect
shared by SKF-525-A. Since evidence which had been obtained in previous
studies indicated that a decrease in metabolism resulted in a decreased
toxicity, a possible explanation for this increase in toxicity could come
from an action of PTU on some other organ or enzyme system which increases
the animals susceptibility to Heptachlor or its epoxide. It is also
possible that a pathway of metabolism of Heptachlor which has not yet
been described may be effected. There is no experimental evidence,
however, to support either possibility.
CHAPTER V
LOCALIZATION OF THE SITE OF ACTION OF HEPTACHLOR
Review of the Literature
There are no reports in the literature concerning pharmacological
investigations of Heptachlor. Studies with Aldrin (8) and Dieldrin (9)
have indicated that these compounds produce an apparent potentiation of
acetylcholine, and an increase in the sensitivity of spinal centers to
this compound. However, neither a direct peripheral effect nor inhibi-
tion of cholinesterase could be demonstrated for these two cyclodiene
insecticides. From these studies, it was concluded that the parasympa-
thetic activity as well as the convulsant properties of Aldrin and
Dieldrin are of central nervous system origin.
Since the principal pharmacologic effects produced by Heptachlor,
and other members of the cyclodiene family, seem to consist chiefly of
stimulation of the central and autonomic nervous systems, pharmacologic
investigation of this compound was confined to those areas. Localization
of the principal site of activity in the central nervous system and the
nature of the autonomic stimulation were the objects of this phase of
the investigation.
59
60
Experimental
Peripheral Effects
The Effect of Heptachlor on Blood Pressure and Respiration
Materials and Methods
The effect of Heptachlor on blood pressure and respiration was
tested on rabbits, cats, and dogs in the following manner:
Blood pressure measurements were taken from a polyethylene
cannula introduced into the right femoral artery. The system used for
these measurements consisted of a Sanborn electromanometer model 121-B-
100, a Sanborn DC amplifier model 64-300B and a Sanborn Twin-Viso
recorder model 60-1300B. The femoral vein of the opposite leg was
cannulated to provide a site for intravenous injection of Heptachlor or
other compounds. All animals were anesthetized with pentobarbital sodium,
35 mg/Kg intraperitoneally and were given additional pentobarbital
intravenously as needed.
The amplitude and frequency of respiration, recorded only for
dogs, was accomplished by using a displacement transducer, attached by
means of a silk suture to the skin of the chest, a few centimeters above
the xyphoid process. A Sanborn strain gauge amplifier, model 64-500B
and a Twin-Viso recorder comprised the remainder of this system.
Heptachlor emulsion, 40 mg/ml was given in 1 ml doses initially,
and in some experiments in increasing amounts up to 10 ml until signs
of central nervous system stimulation appeared. When tremors or
convulsions developed, pentobarbital sodium was given intravenously until
they were controlled.
See Appendix II for the method used to prepare the emulsion.
61
The dogs used in these experiments were healthy 8 to 12 Kg
mongrels. Healthy alley cats weighing 2 to 4 Kg, and white albino
rabbits which weighed 2 to 3 Kg were also used in these studies.
Results
Heptachlor, given in amount sufficient to produce tremors or
convulsions, produced no perceptible change in the blood pressure in
the four rabbits tested. The total amount of Heptachlor necessary to
produce these symptoms was dependent on the depth of anesthesia of the
animal being tested and varied from four injections of 40 mg, spaced at
two-minute intervals, to nine such injections. In trials on three
unanesthetized rabbits, convulsions, terminating in death, were produced
by a single injection of 40 mg of Heptachlor. Convulsions in anesth-
etized animals were easily controlled by cautiously injecting pento-
barbital sodium intravenously until the convulsions or tremors ceased.
This was usually accomplished by 1 to 2 ml of a 25 mg/ml solution of
pentobarbital.
The initial blood pressure studies on cats showed that a profound
hypotensive effect was produced by the Heptachlor emulsion; however, the
control emulsion produced a similar drop in blood pressure. This fall
in pressure was found to be due to a constituent of the lecithin used
to prepare the emulsion. This constituent may be removed by a series
of extraction with organic solvents and the success of this extraction
procedure may be demonstrated by the failure of the purified product to
The method of preparation of the control emulsion is found in Appendix
62
produce a fall in the blood pressure of a barbitalized cat (46). Due to
the expense and complicated nature of this procedure, purification of
the lecithin used in our emulsions was not attempted and blood pressure
studies in the cat were discontinued.
Dogs normally did not show a fall in blood pressure after injec-
tions with emulsions containing lecithin. All dogs used in these
experiments were given 10 ml of the control emulsion intravenously at
the beginning of the experiment to determine if they would react to the
hypotensive constituent of lecithin. Of eight animals tested only one
showed the drop in blood pressure characteristic of the effect produced
in cats. The recording of this blood pressure effect is found in
Figure 10. This animal was not used for blood pressure and respiration
studies.
Heptachlor induced no change in the blood pressure of dogs, when
given in an amount producing symptoms of central nervous system
stimulation. In some instances, however, a fall in pressure did develop
when convulsions were allowed to continue for a few minutes. This fall
was probably secondary to the convulsions rather than a direct effect of
Heptachlor since a pressure drop did not precede any convulsive episode,
but was frequently seen if the convulsions were allowed to continue for
a period of time. The transitory drop seen in Figure 11 could be
duplicated by injection of a similar volume of the control emulsion.
The intravenous administration of 400 mg of Heptachlor produced
a sustained increase in respiratory rate in all animals tested. After
a short delay, most animals began rapid, shallow breathing, which
gradually increased in depth while maintaining the increased frequency.
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A second injection produced an additional increase in respiration which
followed a similar pattern. In deeply anesthetized animals the first
injection did not produce a rapid shallow breathing, but groups of two
to three respirations which occurred at about the same frequency as the
single respiration before Heptachlor was injected. Subsequent injections
produced the type of rapid shallow breathing previously described. These
effects are shown in Figure 11. Respiratory stimulation persisted and
in most cases the animal developed tremors, f asciculations , and convul-
sions. The latter effects may easily be controlled with an intravenous
injection of pentobarbital 25 mg/ml, administered cautiously. However,
this procedure must be repeated frequently as the stimulating effects of
Heptachlor may appear again after five to ten minutes. Attempts to lower
the respiratory rate with pentobarbital in the two cases attempted ended
in death of the animals, before the original rate of respiration had been
reached. Attempts to restore respiration in these animals by the immediate
injection of 400 mg of Heptachlor were unsuccessful.
These experiments indicate that Heptachlor shares the respiratory
stimulating properties of other members of the cyclodiene group, but
lacks the vasodepressive and bradycardia producing effects produced by
Aldrin and Dieldrin.
The Action of Heptachlor on Isolated Rabbit Ileum
Materials and Methods
To determine if a parasympathetic response to Heptachlor could be
demonstrated in smooth muscle, sections of ileum, 2 to 3 cm long, taken
from a site proximal to the cecum were removed from rabbits freshly
66
killed by a blow on the head. This area of the intestine is recommended
by Ludeuna (47, p 142), who states that motility and durability decrease
from the cecum to the duodenal end of the small intestine. The intestine
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strips were placed in warm Ringer's solution at 35 to 38 C, and
constantly aerated. Activity was recorded by means of a displacement
transducer connected to a Sanborn DC amplifier and Twin-Viso recorder,
as previously described for the recording of respiration. When amplitude
and frequency of contraction became stabilized, 1 ml of the control
emulsion was introduced into the bath and allowed to remain in contact
with the intestinal strip for five minutes. Immediately following this
period, 1 ml of the Heptachlor emulsion containing 40 mg/ml was added
to the bath and allowed to remain in contact with the intestinal strip
for five to thirty minutes. At this time, all of the solution was
drained from the bath and replaced with fresh, aerated solution. The
preparation was then tested for its ability to respond to acetylcholine
by adding enough of the latter compound to the bath, to achieve a final
concentration of 1 ppm.
Results
Amplitude, frequency and tone level of these preparations were
not altered by contact with Heptachlor for periods of time up to thirty
minutes. All strips were found to be reactive to acetylcholine following
exposure to Heptachlor. A typical recording obtained in this procedure
is found in Figure 12.
67
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Isolated Lung Perfusion
Materials and Methods
In an effort to determine what part, if any, of the respiratory
difficulty exhibited by animals acutely poisoned with Heptachlor was due
to bronchiolar constriction, isolated rabbit lungs were perfused, using
the method of Sollmann and Von Oettingen (48), described as follows:
A Mariotte bottle filled with Locke-Ringer's solution was connected by
means of a rubber tube to a Woulff bottle, also filled with Locke-Ringer's
solution and maintained at 40 C with a water bath. The outflow of the
Woulff bottle was attached by means of a short rubber tube to a "T"
tube. One free end of the "T" tube was connected by means of a short
piece of rubber tube to a cannula tied into the trachea, and the other
end to a short section of rubber tubing which was occluded by means of
a pinch clamp.
After expelling the air from the lungs, the level of the Mariotte
bottle was adjusted so that air entered the bottle at a rate of about
twenty bubbles per minute. When this rate appeared to be stabilized,
the preparation was considered ready for use. To determine the effect
of Heptachlor on the perfusion rate of this preparation, 1 ml of Heptachlor
emulsion containing 40 mg of Heptachlor was injected into the rubber
tubing, just above the trachea. Immediately following this, 1 ml of
perfusion fluid was removed from the tube connecting the Mariotte
bottle to the Woulff bottle in order to maintain a constant perfusion
pressure. The rate of bubbles entering the Mariotte bottle per minute
were counted every two minutes, for a total of ten times. At this time
1 ml of pilocarpine nitrate, 1:1000 solution, was introduced into the
69
trachea as previously described, and after five minutes the inflow of
bubbles again recorded for one minute to determine if the preparation
would respond to an agent known to produce bronchial constriction.
Prior to injection of Heptachlor emulsion, an injection of the control
emulsion was made to determine if the perfusion rate could be changed
by the emulsion alone.
Results
The rate of perfusion was not altered by either the control or
Heptachlor emulsion in the four preparations tested. Pilocarpine nitrate
tested on the same lungs produced an average drop in perfusion rate of
above 50 per cent. Data obtained from these experiments indicate that
Heptachlor has no direct effect on the bronchial muscles and that
respiratory difficulties observed in acutely poisoned animals are probably
not of a peripheral nature.
Effect on the Frog Rectus Muscle
Materials and Methods
The purpose of this experiment was threefold: 1) to determine if
Heptachlor had a direct effect on skeletal muscle; 2) to determine if
Heptachlor would influence the response of skeletal muscle to acetyl-
choline; and 3) to determine if Heptachlor could inhibit cholinesterase
in vitro .
The rectus abdominus muscle of the frog was removed and set up
for recording of contractions according to the method of Burn (49, p 1).
Contractions were recorded using a displacement transducer and Sanborn
Twin-Viso recorder as previously described for smooth muscle. For parts
70
1 and 2, contraction was obtained by adding 1 ppm acetylcholine to the
bath in which the muscle was immersed. This drug was allowed to act on
the muscle for ninety seconds and the height of contraction recorded on
a stationary drum. At this time the solution was drained from the bath
and fresh frog Ringer's solution was added. When the muscle had returned
to its original length, the drum was advanced 0.5 cm and stopped again
for the next recording. The preparation was allowed to rest five minutes,
at which time 40 mg of Heptachlor, contained in 1 ml of the emulsion was
added. This emulsion was allowed to remain in contact with the muscle
for a period of five minutes and at this time acetylcholine, 1 ppm, was
added and allowed to act on the muscle for ninety seconds as previously
described.
To determine if cholinesterase could be inhibited by Heptachlor
(Part 3), 0.2 ml of horse serum, containing this enzyme was incubated
for five minutes with 1 ml of 1:100,000 acetylcholine solution as a
control. The same procedure was followed after a ten-minute period of
incubation of the horse serum with 1 ml of Heptachlor, 40 mg/ml. Control
contractions produced by 1 ml of 1:100,000 acetylcholine solution added
to the bath were recorded, initially and following the addition of the
last test solution to the bath, to indicate the responsive state of the
muscle. The solutions containing the acetylcholine and the serum
mixtures, were allowed to react with the muscle for ninety seconds and
the bath was drained and fresh frog Ringer's solution added, immediately
following each test. The muscle was allowed five minutes to recover
before the next test was performed. The order in which the tests were
performed was: 1) acetylcholine, 2) acetylcholine plus horse serum,
71
3) horse serum containing Heptachlor plus acetylcholine, and 4) acetyl-
choline. A new muscle was used for each series.
Results
In the four rectus abdominus preparations tested, Heptachlor
showed neither direct action on the muscle, nor potentiation of the
response to a standard concentration of acetylcholine.
Cholinesterase of horse serum was not affected by incubation with
Heptachlor, and no response was obtained when the horse-serum-Heptachlor-
acetylcholine mixture was added to the muscle bath. Inactivation of
acetylcholine was complete in the mixture containing horse serum and
Heptachlor, and in the mixture containing horse serum alone.
Central Effects
Localization of the Site of Action in the Central Nervous System of
the Frog
Materials and Methods
For beginning studies, the frog is an ideal experimental system,
since discrete areas of its central nervous system may be removed quickly
and easily with the preparation surviving for several hours after the
operation.
Decerebration is performed by severing the head of the frog with
a pair of scissors at the level of the posterior margin of the eyes. In
order to remove the optic lobes, the part corresponding to the tectum of
the midbrain in higher animals, decerebration was first performed as
Frogs used in these studies were Rana pipiens , obtained from J. R.
Schettle, Stillwater, Minnesota.
72
above, and the optic Lobes, exposed by this procedure, were removed with
a blunt probe. A third type of preparation produced by removing the
entire brain was obtained by severing the head with scissors just caudal
to the posterior border of the tympanic membrane, resulting in a spinal
animal (50, p 256).
This experiment was performed in two parts: 1) determination
of the level of transection of the brain necessary to prevent development
of convulsion and 2) determination of the level of transection of the
brain necessary to abolish convulsions, once they had developed. For
Part 1, four groups of three frogs each were placed in battery jars
each containing a small amount of frog Ringer's solution. Groups of
three decerebrate, optic lobectomized, and spinal frogs were prepared
as previously described, the remaining group of three frogs served as
unoperated controls. After about fifteen minutes, each frog was given
25 mg of Heptachlor in corn oil by injection into the ventral lymph sac.
These frogs were then observed for twelve hours for appearance of
convulsions.
The procedure for Part 2 was essentially the same as Part 1
except that the operations were not performed until convulsions began
to develop. Frogs were divided into four groups of three frogs each
and given 25 mg of Heptachlor in corn oil by injection into the ventral
lymph sac. The frogs were then placed in battery jars which were
labled as to the type of operation which would be performed in the event
that convulsions did develop. The frogs were observed for a period of
twelve hours and the appropriate operation performed when indicated.
73
Results
The development of convulsions in normal frogs required from six
to ten hours, and consisted of a series of clonic convulsions terminating
in a tonic convulsion of a few seconds duration. This pattern was
repeated following periods during which the frog appeared normal. The
same activity was present in decerebrate and optic lobectomized frogs,
but not in spinal preparations.
The abolition of convulsions in frogs was successful only in the
spinal preparation, but, in general, this procedure was not as successful
as the previous experiment. Since the appearance of convulsions develop
over a long period of time, some operations were not performed until the
symptoms were well advanced, and some of these animals did not recover
from their surgical treatment. Observations on the successful preparations,
however, showed that the convulsions could be stopped by sectioning below
the brain stem, but not by removal of the cerebrum or optic lobes. These
experiments indicate that the principal site of action of Heptachlor in
the frog is in the midbrain.
Localization of the Site of Action in the Central Nervous System of Rats
Materials and Methods
In an effort to determine the principal site of convulsive
activity of Heptachlor in warm-blooded animals, a series of decerebrate-
spinal rats was prepared, using a modification of the separate methods
of decerebration and production of spinal animals of D' Amour and Blood
(44, p 53, 54) as described below.
One day prior to use in the site of action studies, the rats
were anesthetized with chloral hydrate, 400 mg/Kg, and the spinal cord
74
exposed between the sixth and ninth thoracic vertebra. Tension was
applied to the tail, and the cord transected with a scapel„ The cord
was checked for completeness of section and the incision closed with
silk suture and Michael clips. Spinal section at this level does not
interfere with respiration.
The same animal was then prepared for decerebration by making a
mid-line incision over the region of the cerebellum. A hole was drilled
just caudal to the transverse sinus, until the dura could be seen. This
hole was then filled with bone wax and the incision closed with Michael
clips.
A third operative procedure performed at the same time consisted
of disecting the trachea free from overlying muscle and surrounding
fascia, so that a tracheal cannula could later be inserted in a short
period of time.
These procedures resulted in an animal which could be decerebrated,
and in which a tracheal cannula could be installed in less than two
minutes. In addition, spinal reflex activity had returned to the areas
below the section on the day following the operation and the animal was
suitable for the demonstration of the effects of drugs on spinal activity.
On the day following the preparatory operations the animal was
lightly anesthetized with ether and the clips were removed from the
head, exposing the hole caudal to the transverse sinus. A blunt probe
was inserted directly downward and moved from side to side several times
in order to insure complete decerebration. Sectioning at this level is
said to separate all connection rostral to the pons (44, p 54). The
animals were turned on their backs, and the trachea rapidly exposed and
cannulated, using polyethylene tubing of appropriate size.
75
The animals were then observed for the appearance of decerebrate
rigidity which appeared, in successful experiments, in five to fifteen
minutes. This was comprised chiefly of extensor rigidity of the fore-
limbs, as the hindlimbs were previously severed from their cerebral
connections by spinal sectioning.
When the animal's respiration appeared to be stabilized, a test
dose of 0.5 ml of the control emulsion, was administered via the
sublingual vein, and the animal was observed for effects produced by the
emulsion alone. After two minutes, a dose of 0.5 ml of the Heptachlor
emulsion containing 20 mg of Heptachlor was given in a similar manner.
The response to Heptachlor given under these conditions could
fall into three broad categories:
1) No effect, indicating origin of the convulsive activity lay
in the cerebrum.
2) Convulsive activity in the areas still innervated by the
brain stem and upper spinal cord, and not in the areas served by the
spinal cord distal to the section, indicating that principal activity
lay in the brain stem.
3) Convulsive activity both above and below the cord section,
indicating direct action of the spinal cord itself, not dependent on
higher centers.
This method has the advantage that the site of action may be
found using a single animal instead of two, as would be required for
separate decerebrate-spinal animals. One serious drawback, however,
is a high number of unsuccessful experiments, since only six out of
twenty attempted preparations developed signs of decerebrate rigidity
76
and were considered acceptable for use in these experiments. In the
other animals, respiration ceased shortly after decerebration and the
animals died if they were not maintained on a respirator pump. These
animals were considered unsatisfactory for use.
Results
Immediately following the injection of Heptachlor, a marked
increase in rate and depth of respiration appeared. After three to five
minutes, the convulsant effects of this compound could be seen through
the decerebrate rigidity already present in the animal. These effects
consisted of tremors, fasciculations and clonic spasms of the muscles
of the upper extremities. Occasionally these spasms would terminate in
a prolonged tonic extension. During intervening periods, the animal
returned to a state of decerebrate rigidity.
There was no increase in tone or spontaneous activity in the
muscles of the hindlimb of the animal indicating an absence of spinal
or direct skeletal muscle effect of Heptachlor;
All animals were dead within one hour after injection of Heptach-
lor. Respiration ceased before cardiac arrest occurred.
From these experiments it can be concluded that in the rat, as
in the frog, the principal site of activity of Heptachlor lies in the
brain stem. It is probable, however, that Heptachlor, as is the case
of other centrally acting compounds, effects other areas of the central
nervous system, and its classification as a brain stem stimulant is
more one of convenience than precise anatomical localization.
77
Discussion
The two pharmacologic properties described for Heptachlor —
central nervous system stimulation and parasympathomimetic activity --
apparently have similar origins. The absence of a response in vivo by
tissues which respond readily to autonomic drugs leads to the conclusion
that direct stimulation of autonomically innervated structures plays no
part in the symptoms of acute Heptachlor poisoning.
No evidence favoring the inhibition of cholinesterase, direct
action on skeletal muscle, or potentiation of acetylcholine by Heptachlor
was obtained on the isolated frog rectus muscle. These findings, coupled
with the observation that increased secretory activity in rats was most
prominent following periods of convulsions indicates that the central
nervous system is the origin of both convulsive and autonomic effects
of Heptachlor. This conclusion was also reached in studies of Dieldrin
(9) and Aldrin (8). However, these compounds were reported to have
produced bradycardia, vasodepression and potentiation of acetylcholine.
None of these effects were seen with Heptachlor.
Respiratory stimulation was the most consistent effect produced
by Heptachlor in normal-anesthetized and decerebrate animals. This
effect became apparent immediately following the intravenous injection
of large doses of this compound. The cumulative nature of this compound
is demonstrated by the appearance of symptoms after a series of
injections which, individually, would not provoke a response. Respir-
atory stimulation could be increased further by additional injections
of Heptachlor. Tremors and convulsions which usually followed the
initial respiratory stimulation were easily controlled by intravenous
78
pentobarbital. However, respiratory rate was more difficult to control.
The deaths of two animals which resulted during attempts to restore the
respiratory rate to normal suggest that efforts to antidote Heptachlor
by intravenous barbiturates must be made with extreme caution. This
sudden cessation of respiration in animals which had been hyperventilat-
ing as a result of the stimulating effects of Heptachlor, may be due to
a sudden removal of this stimulation by the barbiturates, leaving a state
of fatigue brought on by the prolonged over-activity. Additional
contributory factors would be a lack of the normal carbon dioxide
stimulating effect, since hyperventilation would be expected to result
in a low partial pressure of circulating carbon dioxide. The blood of
such an animal would also be well oxygenated, and an additional means of
respiratory stimulation would not be available. Respiration arrested in
the manner described was not restored by additional Heptachlor injections.
The principal locus of activity of Heptachlor, in the frog and
rat, is the brain stem. In the frog, removal of the cerebrum failed to
stop or prevent the convulsions following injections of Heptachlor,
while removal of the entire brain successfully blocked the appearance
of convulsions. In these respects, and in the general appearance of
the convulsion, Heptachlor is similar to the brain stem stimulants
such as Metrazole. However, no central nervous stimulant has its effects
confined to a discrete area of the central nervous system, but different
areas of the brain seem to vary in susceptibility to a given dose.
Thus Metrazole, a typical brain stem stimulant, in high doses will also
stimulate the spinal cord. In view of the many interconnecting pathways
of the central nervous system it is impossible to assign discrete areas
79
of the central nervous system as the site of action of Heptachlor, or any
other central nervous system stimulant. The respiratory center of the
medulla seems to be especially sensitive to Heptachlor as the effects of
stimulation are first to appear in this area and are not controlled with
measures used successfully for the control of convulsions and tremors.
Evidence favoring medullary action rather than action of the
carotid sinus as the cause of respiratory effects of Heptachlor, arises
from the absence of inspiratory gasps characterized by compounds such
as lobeline or cyanide which stimulate this area directly (23, p 298).
CHAPTER VI
SUMMARY AND CONCLUSIONS
The LD50 values for male and female rats determined in this
study were 59 and 132 mg/Kg respectively. No rats from either sex
survived the first two weeks of a six-month chronic feeding period,
when fed a diet containing 1 or 0.1 per cent Heptachlor. During the
remainder of this period, female rats proved to be more susceptible to
the lethal effects of the higher concentration of Heptachlor than male
rats.
Significant weight changes were found in the liver of male and
female rats fed 0.01 per cent and 0.001 per cent Heptachlor. A signifi-
cant change in kidney weight was also noted with the higher concentration
in both sexes. An increase in testicle weight was found in male rats
fed 0.01 per cent Heptachlor.
Pathological findings of tissues from the organs of Heptachlor
fed animals consisted chiefly of degenerative changes of the liver.
These changes were much more extensive in female than in male rats.
An endocrine basis for the difference in sex response was found
using castrate and hormone treated animals. Castration delays the
appearance of toxic symptoms of Heptachlor in male rats, while testoste-
rone treated rats developed symptoms more rapidly than other groups.
Estradiol was without apparent effect on the toxicity of Heptachlor in
either sex.
80
81
Evidence suggesting metabolic activation of Heptachlor was
obtained from experiments showing that the appearance of toxic symptoms
of Heptachlor could be accelerated by testosterone pretreatment and
delayed by SKF-525-A, compounds known to speed up and retard respectively,
the rate of biotransformation of a number of compounds.
Propylthiouracil, given in the diet of male and female rats
increases the speed of onset and mortality of Heptachlor in the female
and to a lesser extent, the male.
Heptachlor was shown to have no direct effect on isolated smooth
or skeletal muscle. No potentiation of acetylcholine or inhibition of
cholinesterase could be demonstrated, and blood pressure was not effected.
Heptachlor given intravenously produced a sustained increase in
the respiratory rate in both decerebrate and normal anesthetized animals.
This stimulation appeared before other symptoms of central nervous
system stimulation could be seen.
Muscle tremors and convulsions of Heptachlor poisoned animals are
easily controlled with pentobarbital sodium, but frequent administration
are required, due to the persistance of Heptachlor stimulation. Respira-
tion stopped with pentobarbital in Heptachlor poisoned animals could not
be reinitiated by this compound.
The activity of Heptachlor in frogs and rats was found to be
confined to the central nervous system, with the principal site of action
being the brain stem. This finding coupled with failure of Heptachlor
to produce any peripheral response indicates that both the convulsive
and autonomic effects of Heptachlor are mediated through the central
effects of this compound.
>
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APPENDICES
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APPENDIX I
PREPARATION OF THE CORN OIL SOLUTION OF HEPTACHLOR
Usual organic solvents in which Heptachlor is soluble are either
toxic or have undesirable pharmacologic activity. Since Heptachlor is
insoluble in water, it was necessary to use a vegetable oil as the
solvent for oral administration in the acute toxicity studies. Corn
oil was selected because of its ready availability. The solutions were
prepared by dissolving a carefully weighed amount of Heptachlor in a
small amount of hot corn oil and then diluting to volume. In all cases
the quantity of drug to be given per kilogram of body weight was
present in 5 ml of solution.
83
APPENDIX II
PREPARATION OF THE HEPTACHLOR EMULSION FOR
PARENTERAL AND ISOLATED TISSUE STUDIES
For intravenous administration and isolated tissue studies, the
oil solution of Heptachlor was unsuitable because of its immisciblity
with physiologic media. Organic solvents, for reasons previously
stated, were also unsuitable. To solve this problem, an emulsion
similar to one used for intravenous administration of lipids to humans
(46) was prepared. The formula of this emulsion is listed below:
Corn oil containing 207» w/v Heptachlor 20 ml
Lecithin 1 Gm
Pluronic Ffcs 0.2 Gm
Normal saline 80 ml
To prepare the emulsion, the lecithin was dissolved in the corn oil
solution of Heptachlor, and this solution was slowly stirred into the
saline in which the Pluronic Fgg had previously been dissolved, The
resulting crude emulsion was then passed five times through a Manton-
Gaulin two-stage laboratory homogenizer at a pressure of 2500 pounds
per square inch. This procedure is stated to result in a very stable
emulsion, miscible with physiologic media, having a particle size of
one micron or less (46) and containing 40 mg/ml of Heptachlor. A
control emulsion containing 20 per cent pure corn oil was prepared in
a similar manner.
84
APPENDIX III
CALCULATION OF THE LD 50 BY THE LITCHFIELD
AND WILCOXON METHOD
Calculation of the LD^q by this method is accomplished by first
plotting all responses exclusive of the and 100 per cent effects on
log probability paper (Codex 3128). A line is then fitted to these
points by inspection. The corrected and 100 per cent effects are
obtained using information obtained from the graph and a nomograph
o
contained in the original article. A (Chi) test is then performed to
determine if the line fits the data. This method also provides a means
for the determination of the 95 per cent confidence limits of the LD 50
and the slope of the line. The original paper presenting this method
of calculation contains several nomographs and tables which greatly
simplify the calculations (21).
Male Rats
Total contribution of (Chi) 2 from Table 5 - 0.335
Total number of animals - 48
Number of doses ■ K ■ 6
Animals/dose ■ 48/6 - 8
(Chi) 2 - 0.335 x 8 - 2.680
Degrees of Freedom - N - K-2 - 4
(Chi) 2 for N - 4 is 9.49. Since 2.680 is less than 9.49, the data are
not significantly heterogenous and the line is a good fit.
85
86
From the graph of LD.q the following values were found:
LD 84 - 84 mg/Kg
LD 5Q - 59 mg/Kg
LD,, - 42 mg/Kg
Calculation of the Slope Function S
_ LDo./LD^ + LD^/LD
84' 50 ""50' ""16 « 1.415
2
Calculation of the confidence limits of the LD 5Q for 19/20 probability
limits
LDc n x F T _ ■ upper limit
:>U LU 5Q
^50 / F LD 50 " lower limit
r u> R -s 2 - 77 /VF
50
N* ■ total number of animals tested at those doses whose expected
effects were between 16 and 84 per cent » 24
Fld - 1.415 2 ' 777 VK- 1.2
50
LD 5Q x Fjjj - 59.0 x 1.2 - 70.8 mg/Kg
LD 50 / F^ - 59.0 / 1.2-49.1 mg/Kg
Summary
LD 50 = 59(70.8 to 49.1) mg/Kg
S = 1.415(1.14 to 1.175)
Female Rats
The data obtained from the determination of the LDcq are given
in Table 6. Calculations were carried out in the manner previously
described, and only the results are given. The graph for these data
is found in Figure 8.
87
Total number of animals ■ 48
Number of doses ■ K • 6
Animals/dose ■ 8
Total contribution to (Chi) 2 from Table 6 - 0.625
(Chi) 2 for 6 degrees of freedom «= 12.6. Since 5.0 is less than 12.6,
the line is a good fit.
The following values were obtained from the graph:
LD - 195 mg/Kg
84
LD - 132 mg/Kg
LD,, - 95 mg/Kg
The calculated 19/20 probability limits for the slope function were:
upper limit ■ 1.85
lower limit - 1.17
The calculated 19/20 probability limits for the LD 5Q were:
upper limit ■ 154
lower limit "114
The value of the slope function was found to be 1.475
Summary of data:
LD 5Q - 132(114 to 154) mg/Kg
S - 1.475(1.85 to 1.17)
BIBLIOGRAPHY
1. Smyth, Henry P., Jr., Agric. Food Chem. , 4:644 (1956).
2. Frear, Donald E. H. , "Chemistry of the Pesticides," 3rd ed. , D. Van
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Hill Book Co., Inc., New York, N. Y., 1958.
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103:259 (1953).
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43. Leighty, John A., Personal Communication, March 22, 1961.
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in Mammalian Physiology," Rev. ed. , The University of Chicago
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25:692 (1927).
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BIOGRAPHICAL SKETCH
The author was born on May 1, L933 at Holly Springs, Georgia.
He graduated from Canton High School, Canton, Georgia, in 1950 and
entered Mercer University the fall of that year.
He received a B.S. degree in pharmacy from Southern College of
Pharmacy, Atlanta, Georgia, in 1954 Following graduation he served in
the army until August, 1956.
The author began his graduate studies at the University of
Florida during the Fall Semester of 1956, and has been in attendance
at that institution since that time. He expects to receive the Ph.D.
degree in February, 1962.
The author is a member of Kappa Psi pharmaceutical fraternity,
Rho Chi honorary pharmaceutical fraternity, Gamma Sigma Epsilon honorary
chemical fraternity, Phi Sigma honorary biological fraternity and is
an associate member of the American Association for the Advancement of
Science. He has been the recipient of a Graduate Council Fellowship
and is a fellow of the American Foundation for Pharmaceutical Education.
91
This dissertation was prepared under the direction of the Chairman
of the candidate's supervisory committee and has been approved by all
members of that committee. It was submitted to the Dean of the College
of Pharmacy and to the Graduate Council and was approved as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
February 3, 1962
Dean, College of Pharmacy
Dean, Graduate School
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